Genetic engineering of Nannochloropsis oceanica to produce canthaxanthin and ketocarotenoids

Abstract

Background: Canthaxanthin is a ketocarotenoid with high antioxidant activity, and it is primarily produced by microalgae, among which Nannochloropsis oceanica, a marine alga widely used for aquaculture. In the last decade, N. oceanica has become a model organism for oleaginous microalgae to develop sustainable processes to produce biomolecules of interest by exploiting its photosynthetic activity and carbon assimilation properties. N. oceanica can accumulate lipids up to 70% of total dry weight and contains the omega-3 fatty acid eicosapentaenoic acid (EPA) required for both food and feed applications. The genome sequence, other omics data, and synthetic biology tools are available for this species, including an engineered strain called LP-tdTomato, which allows homologous recombination to insert the heterologous genes in a highly transcribed locus in the nucleolus region. Here, N. oceanica was engineered to induce high ketocarotenoid and canthaxanthin production.

Results: We used N. oceanica LP-tdTomato strain as a background to express the key enzyme for ketocarotenoid production, a β-carotene ketolase (CrBKT) from Chlamydomonas reinhardtii. Through the LP-tdTomato strain, the transgene insertion by homologous recombination in a highly transcribed genomic locus can be screened by negative fluorescence. The overexpression of CrBKT in bkt transformants increased the content of carotenoids and ketocarotenoids per cell, respectively, 1.5 and 10-fold, inducing an orange/red color in the bkt cell cultures. Background (LP) and bkt lines productivity were compared at different light intensities from 150 to 1200 µmol m^-2 s^-1: at lower irradiances, the growth kinetics of bkt lines were slower compared to LP, while higher productivity was measured for bkt lines at 1200 µmol m^-2 s^-1. Despite these results, the highest canthaxanthin and ketocarotenoids productivity were obtained upon cultivation at 150 µmol m^-2 s^-1.

Conclusions: Through targeted gene redesign and heterologous transformation, ketocarotenoids and canthaxanthin content were significantly increased, achieving 0.3% and 0.2% dry weight. Canthaxanthin could be produced using CO₂ as the only carbon source at 1.5 mg/L titer. These bkt-engineered lines hold potential for industrial applications in fish or poultry feed sectors, where canthaxanthin and ketocarotenoids are required as pigmentation agents.

Keywords: Nannochloropsis; Canthaxanthin; Carotenoids; Ketocarotenoids; Microalgae.

Fig. 1

CrBKT expression in Nannochloropsis oceanica. (a) Schematic representation of the carotenoid pathway towards astaxanthin biosynthesis (b) Schematic diagram of the expression vector used to transform N. oceanica LP background strain

Fig. 2

Screening of Nannochloropsis oceanica lines expressing CrBKT. (a) Fluorescence screening for the presence of tdTomato to select positive transformant candidates. (b) PCR on bkt-transformants to confirm correct insertion of the BKT construct in LP strain. (c) Spectra of acetone extracts from LP background and bkt- transformants. (d) LP and bkt transformants grown at 40 µmol m^− 2s^− 1 in ASW medium

Fig. 3

HPLC chromatogram of pigment extracts of LP and bkt strains. 1: violaxanthin, 2: astaxanthin, 3 vaucheriaxanthin, 4 antheraxanthin, 5 vaucheriaxanthin ester, 6 zeaxanthin, 7 canthaxanthin, 8 chlorophyll a, 9 β-carotene, 10 adonirubin, 11 adonixanthin. The different peaks were identified by comparing retention times and spectra to commercially available standards (CaroteNature GmbH, Munsingen, Switzerland)

Fig. 4

Photosynthetic parameters measured in LP and bkt lines. (a) Photosystem II Photochemical quantum yield (Y(II)), (b) Electron Transport Rate (ETR), (c) Non-photochemical quenching (NPQ) and (d) photochemical quenching (qP) were measured at different actinic lights on dark adapted cells. Data are expressed as means ± standard deviation (n = 3). Significantly different values are indicated with *

Fig. 5

Biomass accumulation in LP and bkt lines at different light intensities. Growth curve of LP (a) and bkt (b) strain in airlifted photobioreactors at 150, 600 and 1200 µmol m^− 2s^− 1. Growth curves were obtained monitoring OD at 720 nm (c) Representative pictures at the end of the growth. Biomass productivities (d) and maximal productivity (e) obtained from LP and bkt grown as reported in panels a and b. Data are expressed as means ± standard deviation (n = 3). Significantly different values are indicated with different letters

Fig. 6

Carotenoids and ketocarotenoids productivities in LP and bkt lines. Total carotenoids, ketocarotenoids and canthaxanthin content per dry weight (a) and average daily productivity (b) at the end of the experiment described in Fig. 5. Data are expressed as means ± standard deviation (n = 3). Significantly different values are indicated with different letters

Fig. 7

Ketocarotenoids and canthaxanthin productivity during the growth curve. Volumetric production (a) and volumetric productivity (b) correlated with optical density measurements at 720 nm during the growth 150 µmol m^− 2s^− 1. Data are expressed as means ± standard deviation (n = 3)